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The Journal of Immunology, 2004, 173: 5697-5703.
Copyright © 2004 by The American Association of Immunologists

Rac1 Contributes to Maximal Activation of STAT1 and STAT3 in IFN-{gamma}-Stimulated Rat Astrocytes1

Eun Jung Park2,*, Kyung-Ae Ji2,{dagger}, Sae-Bom Jeon{dagger},{ddagger}, Woo-Hyuck Choi{dagger}, Inn-oc Han§, Hye-Jin You, Jae-Hong Kim, Ilo Jou* and Eun-Hye Joe3,*,{dagger},{ddagger}

* Department of Pharmacology, {dagger} Department of Neuroscience, {ddagger} Brain Disease Research Center, School of Medicine, Ajou University, Suwon, Korea; § Research Institute, National Cancer Center, Goyang, Gyeonggi, Korea; and School of Life Sciences and Biotechnology, Korea University, Seoul, Korea


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Rac1 GTPase is implicated as a signaling mediator in various cellular events. In this study, we show that Rac1 contributes to IFN-{gamma}-induced inflammatory responses in rat astrocytes. We revealed that IFN-{gamma} rapidly stimulated activation of Rac1 in C6 astroglioma cells by investigating GST-PAK-PBD-binding ability. We also found that Rac1 deficiency led to attenuation of IFN-{gamma}-responsive transcriptional responses. Compared with levels in control cells, IFN-{gamma}-induced IFN-{gamma}-activated sequence promoter activity was markedly reduced in both C6 astroglioma cells and primary astrocytes expressing RacN17, a well-characterized Rac1-negative mutant. The expression of several IFN-{gamma}-responsive genes, such as MCP-1 and ICAM-1, was also reduced in cells expressing RacN17. Consistent with these observations, IFN-{gamma}-induced phosphorylation of STAT1 and STAT3 was lower in C6 cells expressing RacN17 (referred to as C6-RacN17) than in control cells. However, there was no difference in expression level of IFN-{gamma}R{alpha} subunit and IFN-{gamma}-induced phosphorylation of JAK1 between C6 control and C6-RacN17 cells. Interestingly, Rac1 appeared to associate with IFN-{gamma}R{alpha} and augment the interaction of IFN-{gamma}R with either STAT1 or STAT3 in response to IFN-{gamma}. Taken together, we suggest that Rac1 may serve as an auxiliary mediator of IFN-{gamma}-signaling, at least at the level of STAT activation, thus contributing to maximal activation of IFN-{gamma}-responsive inflammatory signaling in rat astrocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Brain inflammation is closely associated with the pathogenesis of various neurodegenerative diseases. Compared with inflammation in peripheral tissues, inflammation in brain appears to follow distinct pathways and time-courses. The major immune cells that respond to inflammatory stimuli in the brain are astrocytes and microglia. The inflammatory responses in these cells are coordinated by the subsequent production of cytokines, chemokines, and reactive oxygen species (1, 2, 3). These molecules function in synergistic and/or antagonistic manner, eventually leading to neurodegeneration via inflammatory cascade. Thus, it is important to understand the molecular mechanisms governing immune reactions in the brain.

IFN-{gamma} is an inflammatory cytokine that exhibits antiviral, antimicrobial, antitumor, and immunomodulatory properties (for reviews, see Refs. 4, 5). In the brain, IFN-{gamma} can induce reactive astrogliosis. Upon exposure to IFN-{gamma}, astrocytes proliferate and increase expression of various inflammation-associated molecules, including ICAM-1 and TNF-{alpha}, as well as glial fibrillary acidic protein (6, 7, 8). Generally, binding of IFN-{gamma} to its receptors induces assembly of an active receptor complex, and consequent transphosphorylation of the receptor-associated JAK1 and JAK2 (4, 5). Phosphorylated JAK1 and JAK2 lead to phosphorylation of tyrosine residues in the cytoplasmic tail of the receptor, which provide the docking sites for STAT. After being recruited to the receptor complex, STAT becomes phosphorylated on both Tyr701 and Ser727. Phosphorylated STAT is released from the receptor complex and forms dimers. These dimers translocate to the nucleus where they directly bind to IFN-{gamma}-activated sequences (GAS), 4 thereby regulating transcription of IFN-{gamma}-responsive genes (9, 10).

There are increasing reports that JAK/STAT signaling is finely regulated by cross-talk and convergence between different signaling pathways. G protein-coupled receptors (GPCR) have been shown to closely associate with JAK/STAT signaling (11, 12, 13). For instance, GPCR agonist angiotensin II can stimulate the tyrosine phosphorylation of JAKs and STAT by angiotensin II AT1 receptor (11). Recently, it has been reported that small GTPase Rho and Rac are necessary for GPCR-induced activation of JAK and STAT (14). In line with these findings, several reports have shown that Rac1 directly interacts with STAT3 and stimulates the phosphorylation of STAT3 in response to growth factors (15, 16).

In immune responses, functional importance of Rac has been emerging (17, 18, 19). Studies by Rac-deficient mice and patients showed that Rac is closely associated with immunodeficiency syndrome and abnormalities in host defense against pathogenic infection (20, 21). Based on these evidences, we questioned whether Rac1 could contribute to the activation of JAK/STAT pathways triggered by IFN-{gamma} in rat astrocytes. In this study, we show that IFN-{gamma} rapidly activates Rac1, and that Rac1 deficiency leads to attenuation of STAT activation and subsequent inflammatory responses by IFN-{gamma}. The results in this study suggest that Rac1 may participate as an auxiliary component of IFN-{gamma}-induced inflammatory signaling in rat brain astrocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Rat IFN-{gamma} and Ab against phosphorylated JAK1 were from Calbiochem (San Diego, CA). MEM, Lipofectamine Plus, and G418 antibiotics were from Invitrogen Life Technologies (Carlsbad, CA. DMEM and FBS were from HyClone (Logan, UT). Abs against STAT1, Tyr701-phosphorylated STAT1, and STAT3 were from Cell Signaling Technology (Beverly, MA). Abs against JAK2, phosphorylated JAK2, and rac1 were from Upstate Biotechnology (Lake Placid, NY). Ab against IFN-{gamma}R{alpha} was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Peroxidase-conjugated secondary Abs were from Vector Laboratories (Burlingame, CA).

Cell culture

C6 rat astroglioma cells were obtained from the American Type Culture Collection (Manassas, VA; ATCC CCL-107). Cells were grown in DMEM supplemented with 10% (v/v) FBS. Primary astrocytes were cultured from the cerebral cortices of 1- to 3-day-old Sprague Dawley rats as previously described (22). Briefly, the cortices were triturated into single cells in MEM containing 10% FBS and plated into 75 cm2 T-flasks (0.5 hemisphere/flask) for 2–3 wk. To prepare pure astrocytes, microglia were detached from the flasks by mild shaking and the cells remaining in the flask were harvested with 0.1% trypsin. Astrocytes were plated in dishes and cultured in MEM supplemented with 10% FBS.

GST-PAK-PBD-binding assays

The activation of Rac1 was determined as previously reported (23). Briefly, the pGEX construct encoding the GTPase-binding domain of PAK1 was expressed in Escherichia coli as a GST fusion protein (GST-PBD). C6 cells were incubated in serum-free medium for 24 h, and then treated with 10 U/ml IFN-{gamma}. Cells were lysed and incubated with 3 µg of GST-PBD. To detect GTP-bound Rac1, proteins were separated by SDS-PAGE, and Western blot analysis was performed using Abs against Rac1.

Plasmids

Constitutively active mutant, and pEXV-RacN17, a dominant-negative mutant of Rac1, were gifts from Dr. A. Hall (University College London, London, U.K.). The 8-GAS luciferase reporter construct was a gift from Dr. M.-h. Shong (Chungnam National University, Daejon, Korea).

Generation of cells stably expressing RacN17

For the generation of C6 clonal cell lines stably expressing RacN17, the pEXV-RacN17 and pcDNA-NeoR plasmid constructs were cotransfected into C6 cells by Lipofectamine Plus. The transfected cells were selected and maintained in the presence of 400 µg/ml G418 antibiotic in DMEM supplemented with 10% FBS. Individual G418-resistant colonies were isolated 2–3 wk later and expanded into cell lines. Expression of RacN17 protein in the clones was confirmed by Western blot analysis using an anti-c-Myc epitope Ab (24).

Luciferase assay

Transient transfections were performed in duplicate on 35-mm dishes using Lipofectamine Plus reagents as instructed by the manufacturer (Invitrogen Life Technologies). To normalize the variations in cell number and transfection efficiency, all clones were cotransfected with pCMV-{beta}-galactosidase for 24 h. Luciferase assay was performed according to the manufacturer’s instruction (Promega, Madison, WI). Luciferase activity was measured using 20 µl of cell extract in 100 µl of assay buffer. The light intensity was measured for 30 s on a luminometer (Berthold Lumat LB9501; Berthold Technologies, Oak Ridge, TN). Luciferase activity was normalized by measurement of the {beta}-galactosidase activity (in OD420).

Western blot analysis

Cells were washed twice with cold PBS, and then lysed in ice-cold modified RIPA buffer (50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM Na3VO4, and 1 mM NaF) containing protease inhibitors (2 mM PMSF, 100 µg/ml leupeptin, 10 µg/ml pepstatin, 1 µg/ml aprotinin, and 2 mM EDTA). The lysate was centrifuged for 20 min at 13,000 x g at 4°C, and the supernatant was collected. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane. The membrane was incubated with primary Abs, peroxidase-conjugated secondary Abs, and then visualized using an ECL system.

Immunoprecipitation

The cells grown on the 100-mm dishes were washed with PBS twice before lysis. The modified RIPA buffer containing the protease inhibitors were added for cell lysis. The cell lysate was collected, and centrifuged at 13,000 x g for 20 min. The supernatants were incubated for overnight with the primary Abs with rocking, and the immune complexes were collected on protein G beads for 2 h. The immunoprecipitates were washed three times with modified RIPA buffer and collected. The resulting precipitates were then examined by SDS-PAGE and immunoblot analysis.

RT-PCR

Total RNA was extracted using RNAzol B (Tel-Test, Friendswood, TX) and cDNA was prepared using reverse transcriptase that originated from Avian Myeloblastosis Virus (TaKaRa, Shiga, Japan), according to the manufacturer’s instructions. PCR was performed with 30 cycles of sequential reactions: 94°C for 30 s, 55°C for 30 s, and 72°C for 90 s. Oligonucleotide primers were purchased from Bioneer (Seoul, Korea). The sequences of PCR primers were as follows: reverse, 5'-AAGGCCGCAGAGAGCAAAAGAAGC-3', and forward, 5'-CTGGAGAGCACAAACAGCAGAG-3', for ICAM-1; reverse, 5'-ATGCAGGTCTCTGTCACGCT-3', and forward, 5'-CTAGTTCTCTGTCATACTGG-3', for MCP-1; reverse, 5'-AGATCCACAACGGATACATT-3', and forward, 5'-TCCCTCAAGATTGTCAGCAA-3', for GAPDH.

Flow cytometric analysis

Cells were incubated in serum-free medium for 24 h, and then treated with 10 U/ml IFN-{gamma} for the indicated times. The cells were washed twice with PBS containing 1% FBS, collected, and then stained with FITC-conjugated anti-rat CD54 (ICAM-1) mAb (BD Pharmingen, San Diego, CA) for 30 min at 4°C. After washing, the cells were analyzed with a FACSVantage (BD Biosciences, San Jose, CA), and the data were processed using the CellQuest program (BD Biosciences) and WinMDI.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IFN-{gamma} stimulates the activation of Rac1

In an effort to investigate whether Rac1 mediates the action of IFN-{gamma} in astrocytes, we examined whether IFN-{gamma} could stimulate the activation of Rac1 in C6 astroglioma cells. The activation of Rac1 was defined as its binding ability to the GTPase-binding domain of p21-activated kinase (PAK-PBD) (23). C6 cells were stimulated with 10 U/ml IFN-{gamma}, and GST-PAK-PBD-bound active Rac1 was detected by Western blot analysis using anti-Rac1 Ab. Interestingly, binding of Rac1 to GST-PAK-PBD significantly increased within 5 min of IFN-{gamma} treatment, reached the peak at 15–30 min, and then gradually decreased (Fig. 1A, and data not shown). To further examine the IFN-{gamma}-induced activation of Rac1, C6 cells were treated with various concentrations of IFN-{gamma} for 15 min, and then binding ability of Rac1 to GST-PAK-PBD was examined. Binding of Rac1 to GST-PAK-PBD was slightly detected in cells treated with 0.1 U/ml IFN-{gamma}, and clearly observed in cells treated with 1–10 U/ml IFN-{gamma} (Fig. 1B). These results indicate that IFN-{gamma} rapidly stimulates the activation of Rac1, suggesting the possibility that Rac1 may contribute to the function of IFN-{gamma} in astrocytes.



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  		FIGURE 1. IFN-{gamma} induces activation of Rac1. A, C6 cells were incubated in serum-free medium for 24 h and treated with 10 U/ml IFN-{gamma} for the indicated times. The amount of Rac1 bound to GST-PAK1-PBD fusion protein was analyzed by Western blot as described in Materials and Methods. B, C6 cells were treated with various concentration of IFN-{gamma} for 15 min, and then GST-PAK-PBD-bound active Rac1 was detected by Western blot analysis using anti-Rac1 Ab. GST band was shown as a loading control.

 
Dominant-negative Rac1 reduces IFN-{gamma}-induced GAS promoter activity in rat astrocytes

Because IFN-{gamma} rapidly activated Rac1 in astrocytes, we investigated whether Rac1 could participate in IFN-{gamma}-induced signaling. For this, we first examined the effect of Rac1 dominant-negative mutant (pEXV-RacN17) or constantly active mutant (pEXV-RacV12) on transcriptional activation of GAS. C6 cells were transiently transfected with luciferase reporter plasmids containing 8-GAS elements and either pEXV-RacN17 or pEXV-RacV12, or control vector. Twenty-four hours after transfection, the cells were stimulated with 10 U/ml IFN-{gamma} for 24 h. Luciferase activity was then determined and the result was normalized for transfection efficiency by comparing {beta}-galactosidase activity. IFN-{gamma} dramatically enhanced GAS luciferase activity in control cells transfected with vector alone. By contrast, IFN-{gamma}-induced GAS luciferase activity was significantly reduced in cells transfected with RacN17 (Fig. 2A). There was no significant difference in luciferase activity between cells transfected with RacV12 and control vector (Fig. 2A). Intriguingly, the basal level of GAS luciferase activity was also lowered in cells transfected with RacN17 compared with that in cells transfected with control vector (Fig. 2A). To extend upon these studies, C6 cells were transiently transfected with different concentrations of RacN17. Consistent with the above result, relative luciferase activity was diminished with increasing the concentration of RacN17 (Fig. 2B).



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  		FIGURE 2. Expression of RacN17 markedly attenuates GAS promoter activity in rat astrocytes. A, The 8-GAS luciferase construct was cotransfected with pEXV-RacN17, pEXV-RacV12, or vector alone into C6 cells for 24 h. The cells were untreated or treated with 10 U/ml IFN-{gamma} for 24 h and then assayed for luciferase activity. B, C6 cells were cotransfected with the 8-GAS luciferase construct and various concentrations of pEXV-RacN17 for 24 h. Cells were then treated with 10 U/ml IFN-{gamma} for 24 h and harvested for luciferase assay. C, The 8-GAS luciferase construct was cotransfected with pEXV-RacN17 or vector alone into primary rat astrocytes, and the cells were treated with the indicated concentration of IFN-{gamma} for 24 h. The luciferase activities shown in this study were calculated following measurement of {beta}-galactosidase activity to normalize for transfection efficiency. Values are mean ± SD of duplicates. Data shown are representative of at least four independent experiments.

 
We next examined whether the effect of RacN17 on IFN-{gamma}-induced GAS activity was also observed in primary astrocytes. Primary rat astrocytes were cotransfected with RacN17 and GAS luciferase reporter genes, and then treated with various concentrations of IFN-{gamma} for 24 h. In primary astrocytes, as well as C6 glioma cells, the expression of RacN17 significantly reduced IFN-{gamma}-induced GAS luciferase activity (Fig. 2C). These findings support the hypothesis that Rac1 plays a role in IFN-{gamma} signaling in rat astrocytes.

The expression of IFN-{gamma}-responsive genes is attenuated in C6 cells expressing RacN17

To further evaluate the contribution of Rac1 to IFN-{gamma}-induced cellular responses, we established the C6-RacN17 cell lines, dominant-negative mutant cells stably expressing RacN17 (referred to as C6-RacN17) (24). After examining the expression level of RacN17 using anti-c-Myc epitope Ab (Fig. 3A), we first investigated whether IFN-{gamma}-induced GAS luciferase activity was also reduced in C6-RacN17 stable cell lines. Cells were incubated with 10 U/ml IFN-{gamma} for the indicated times, after which luciferase activity was measured. We found that IFN-{gamma}-induced GAS luciferase activities were significantly reduced in C6-RacN17 cells, compared with those in C6 control cells (Fig. 3A). These results are in agreement with the data from C6 cells transiently transfected with RacN17.



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  		FIGURE 3. IFN-{gamma}-enhanced GAS luciferase activity and gene expression are attenuated in cells expressing RacN17. A, The difference of GAS luciferase activity between C6 control cells (a) and C6-RacN17 cells (b) increased in a time-dependent fashion. The expression level of RacN17 was determined by Western blotting using an anti-c-Myc Ab in C6-RacN17 stable clone. C6 and C6-RacN17 cells were treated with 10 U/ml IFN-{gamma} for the indicated times, and then assayed for luciferase activity. B, The transcriptions of MCP-1 and ICAM-1 were low down in the C6-RacN17 cells. Cells were treated with 10 U/ml IFN-{gamma} for the indicated times, and then RT-PCR analysis was performed to detect mRNA expression of MCP-1 and ICAM-1. The expression of GAPDH was detected as an internal control. C, Cell surface expression of ICAM-1 was low in C6-RacN17 cells compared with that in C6 control cells. Cells were treated with 10 U/ml IFN-{gamma} for the indicated times, and then FACS analysis was performed with FITC-conjugated anti-rat CD54 (ICAM-1) mAb. Data shown are representative of more than three independent experiments.

 
The decline of IFN-{gamma}-enhanced GAS promoter activity in Rac1-deficient cells led us to investigate the effect of Rac1 deficiency on the transcript level of IFN-{gamma}-responsive genes. We examined the mRNA levels of MCP-1 and ICAM-1, which are previously reported to have functional GAS elements (25, 26). Cells were stimulated with 10 U/ml IFN-{gamma} for the indicated times, and total RNA was extracted for RT-PCR analysis. As shown in Fig. 3B, IFN-{gamma}-induced transcript levels of MCP-1 and ICAM-1 were relatively low down in C6-RacN17 cells compared with C6 control cells. We observed similar results in primary astrocytes transiently transfected with RacN17 (data not shown).

In addition, we investigated the cell surface expression of ICAM-1 by flow cytometric analysis using FITC-conjugated anti-rat CD54 (ICAM-1) mAb. Consistent with above results, the basal expression level of ICAM-1 was significantly low down in C6-RacN17 cells compared with that in C6 control cells, and IFN-{gamma}-induced enhancement of ICAM-1 expression was also weak in C6-RacN17 cells (Fig. 3C). These results further support the notion that Rac1 contributes to IFN-{gamma}-induced transcriptional activation in astrocytes.

RacN17 does not affect the expression level of IFN-{gamma}R{alpha} subunit

Given that the expression of the dominant-negative Rac1 significantly attenuated GAS-driven gene expression, we questioned whether RacN17 caused down-regulation of IFN-{gamma}R. Using Ab against IFN-{gamma}R{alpha} subunit, we tested this possibility. Western blot analysis showed that the expression level of IFN-{gamma}R{alpha} was not different in C6 control and C6-RacN17 cells (Fig. 4A). In cells transiently expressing RacN17, we did not detect the reduced expression of IFN-{gamma}R{alpha}, compared with cells expressing vector alone (Fig. 4B). We also found that IFN-{gamma} did not change the expression level of IFN-{gamma}R{alpha} in C6 cells and rat primary astrocytes (Fig. 4, A and B, and data not shown). These results indicate that inhibitory effect of dominant-negative Rac1 on IFN-{gamma}-induced transcriptional regulation is not due to down-regulation of IFN-{gamma}R expression.



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  		FIGURE 4. The expression level of IFN-{gamma}R is not changed by expression of RacN17. A, Cells were untreated or treated with 10 U/ml IFN-{gamma} for 12 h, and then assayed for Western blot analysis using Ab against IFN-{gamma}R{alpha}. B, After C6 cells were transiently transfected with pEXV-RacN17 or vector alone for 24 h, the expression level of IFN-{gamma}R{alpha} was assessed by Western blot analysis. Data shown are representative of four independent experiments.

 
Rac1 plays a role in IFN-{gamma}-stimulated phosphorylations of STAT1 and STAT3 but not JAK1

The activation of STAT1 and STAT3 is a prerequisite for GAS promoter activation, and both tyrosine and serine phosphorylation are required for maximal STAT activity (5, 14). Given the critical role of STAT1 and STAT3 in IFN-{gamma} signaling, we examined activation of these molecules in cells expressing RacN17. Cells were stimulated with IFN-{gamma} for the indicated times, and the levels of phosphorylated STAT1 and STAT3 were determined by Western blot analysis using Abs against tyrosine-phosphorylated STAT1 and STAT3 (Fig. 5A). In C6 control cells, strong phosphorylations of STAT1 and STAT3 were observed within 2.5 min of IFN-{gamma} addition, and the levels were sustained up to 1 h. However, IFN-{gamma}-stimulated phosphorylation of STAT1 and STAT3 in C6-RacN17 cells was significantly weak (Fig. 5A). The expression level of total STAT1 and STAT3 was not different in C6 control cells and C6-RacN17 cells. Taken together, these results showed that the activation of STAT1 and STAT3 differed between C6 control and C6-RacN17 cells, which is consistent with the differences in GAS activity and mRNA expression of MCP-1 and ICAM-1 between C6 control and C6-RacN17 cells.



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  		FIGURE 5. Expression of RacN17 attenuates the IFN-{gamma}-induced phosphorylation of STAT1 and STAT3, but not JAK1. A, Cells were serum-starved for 24 h and then stimulated with 10 U/ml IFN-{gamma} for the indicated times. Cell lysates were separated by 10% SDS-PAGE and Western blot analysis was performed using Abs specific for phospho-Tyr-STAT1 (pSTAT1) or phospho-Tyr STAT3 (pSTAT3). The membrane was then stripped and analyzed sequentially with Abs specific for total STAT1 or STAT3, and actin, respectively. B, RacN17 does not affect IFN-{gamma}-induced phosphorylation of JAK1. The phosphorylation of JAK1 was detected by Western blot analysis using Ab specific for phospho-Tyr-JAK1 (pJAK1). The membrane was stripped and probed with actin Ab to show the amount of loaded protein. Ca, C6 cells were treated with 10 U/ml IFN-{gamma} for 2.5 min and 5 min. Cb, C6 cells were pretreated with AG490 (5–10 µM) for 1 h and then stimulated with 10 U/ml IFN-{gamma} for 30 min. The amount of Rac1 bound to GST-PAK-PBD fusion protein was analyzed by Western blot using Ab against Rac1. GST band was shown as a loading control. Data shown are representative of four independent experiments.

 
Next, we investigated whether RacN17 affected the activation of JAK because both phosphorylations of STAT1 and STAT3 depend on the activation of JAK. Cells were stimulated with IFN-{gamma} for the indicated times, and cell lysates were Western blotted using Ab against Tyr-phosphorylated JAK1 (Fig. 5B). Interestingly, in C6-RacN17 cells, JAK1 phosphorylation was not attenuated: in both C6 control cells and C6-RacN17 cells, JAK1 phosphorylation was detected within 2.5 min of IFN-{gamma} addition, and sustained up to 60 min (Fig. 5B). These results suggest that Rac1 deficiency lead to blocking of IFN-{gamma}-signaling at the level below JAK1. To further delineate this notion, we finely examine the kinetics of Rac1 activation in relation to JAK activation. Binding of Rac1 to GST-PAK-PBD was observed at 2.5 min of IFN-{gamma} treatment as rapid as phosphorylation of JAK1 (Fig. 5Ca). Thus, we investigated whether IFN-{gamma}-induced activation of Rac1 is affected by inhibition of JAK activity in C6 cells. In agreement with the above data (Fig. 5B), treatment of AG490, a specific inhibitor of JAK1 and JAK2, significantly diminished the binding of Rac1 to GST-PAK-PBD in C6 cells (Fig. 5Cb). These results indicate that IFN-{gamma} may activate Rac1 through activation of JAK, and Rac1 deficiency has no effect in IFN-{gamma}-induced activation of JAK in astrocytes.

Rac1 associates with IFN-{gamma}R{alpha}, STAT1, and STAT3

Our above results raised the question of how Rac1 could modulate the phosphorylation of STAT1 and STAT3 in astrocytes. In an attempt to understand it, we examined whether Rac1 could interact with IFN-{gamma}R. C6 cells were treated with 10 U/ml IFN-{gamma}, and the cell lysates were immunoprecipitated with IFN-{gamma}R{alpha}. Then, Western blot analysis was performed with Abs against Rac1, JAK1, STAT1, and IFN-{gamma}R{alpha}, respectively. Interestingly, coimmunoprecipitation assay showed that IFN-{gamma}R{alpha} interacted with Rac1 as well as JAK1 and STAT1 (Fig. 6A). Furthermore, the association of IFN-{gamma}R{alpha} with Rac1 was detected in the absence of IFN-{gamma}, while association of IFN-{gamma}R{alpha} with either JAK1 or STAT1 was detected in the presence of IFN-{gamma} (Fig. 6A). To confirm this notion, we performed immunoprecipitation assay with Rac1 Ab. Consistently, the association of Rac1 with IFN-{gamma}R was also detected in immunoprecipitated complex with Rac1 (Fig. 6B). However, we did not observe the difference in the association of Rac1 with IFN-{gamma}R{alpha} in C6-RacN17 cells compared with that in C6 control cells (Fig. 6B). These results suggest that Rac1 may interact with IFN-{gamma}R{alpha}, but their interaction is not regulated by IFN-{gamma}-induced activation of Rac1.



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  		FIGURE 6. Rac1 interacts with IFN-{gamma}R{alpha}, JAK, and STAT. A, C6 cells were incubated in serum-free medium for 24 h and treated with 10 U/ml IFN-{gamma} for the indicated times. Lysates were immunoprecipitated with Ab against IFN-{gamma}R{alpha} subunit, and were subjected to Western blot analysis. The membrane was serially blotted using Abs against JAK1, STAT1, and Rac1. B, C6 and C6-RacN17 cells were incubated in serum-free medium for 24 h and treated with IFN-{gamma} for the indicated times. Lysates were immunoprecipitated with Ab against Rac1, and subjected to Western blot analysis. The membrane was serially blotted using Abs against IFN-{gamma}R{alpha} and Rac1. C, C6 and C6-RacN17 cells were incubated in serum-free medium for 24 h and treated with 10 U/ml IFN-{gamma} for the indicated times. Lysates were immunoprecipitated with Ab against Rac1, and were subjected to Western blot. The membrane was serially blotted using Abs against STAT1, STAT3, and Rac1. Data shown are representative of three independent experiments.

 
Given the report that Rac1 bind to and regulate STAT3 activity in response to epidermal growth factor (15), we investigated whether Rac1 could interact with STAT1 and STAT3 in IFN-{gamma}-treated C6 cells. C6 cells were stimulated with IFN-{gamma} and immunoprecipitated with Rac1 Ab. Then, Western blot analysis was performed with STAT1 or STAT3. As shown in Fig. 6C, both STAT1 and STAT3 were coimmunoprecipitated with Rac1 in C6 cells (Fig. 6C, a and b). Although STAT1 and STAT3 were faintly detected in the absence of IFN-{gamma}, they were clearly detected in the presence of IFN-{gamma} (Fig. 6C, a and b). Thus, we examined whether Rac1 deficiency could affect the IFN-{gamma}-stimulated Rac1 association with STAT1 or STAT3. Interestingly, though we overexposed the film, we did not observe the enhanced association of Rac1 with either STAT1 or STAT3 in the presence of IFN-{gamma} (Fig. 6C, a and c). These results imply that Rac1 may associate with either STAT1 or STAT3 in response to IFN-{gamma}, and their association may contribute to maximal activation of STAT1 and STAT3 in C6 cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Rac GTPase has been shown to play a key regulatory role in various cellular events, such as cytoskeletal organization, cell growth, development, apoptosis, transcriptional regulation, and superoxide production (for reviews, see Refs. 27, 28, 29). Recently, several reports have suggested that Rac GTPase is important in immune responses (17, 18, 19, 20, 21). However, little is known about the specific function of Rac in IFN-{gamma}-induced immune responses. In this study, we show that Rac1 acts as a mediator in IFN-{gamma}-induced activation of STAT inflammatory signaling pathway, thereby contributing to full activation of IFN-{gamma}-induced signaling responses in rat astrocytes.

The involvement of Rac1 in IFN-{gamma}-signaling was obtained from measurement of Rac1 activity. Rac1 is a binary switch that cycles between active GTP-bound and inactive GDP-bound states to regulate various cellular processes. Rac1, in its GTP-bound active forms, has been reported to bind to PAK via interaction with PBD (23). In C6 cells, the binding of Rac1 to the GST-PAK-PBD was significantly enhanced by treatment of IFN-{gamma} (Fig. 1). Guanine nucleotide exchange factors, such as Vav and son of sevenless (Sos)-1 activate Rac1 by exchanging GDP into GTP (30, 31). Several studies have reported the possible involvement of Vav in IFN-{gamma} and -{alpha} signaling. In murine RAW 264.7 macrophages, IFN-{gamma} triggers the rapid tyrosine phosphorylation of Vav (32). In megakaryocytic cells, Vav is physically associated with both {alpha} and {beta} IFN-{gamma}R chains (33). In addition, receptor tyrosine kinases activate Sos-1 through interaction with the adaptor molecule, Grb2 (34), though it has not been clearly defined whether Sos-1 is activated by IFN-{gamma}. In our experiments, activation of Rac1 was abolished in the presence of JAK inhibitor, AG490, while Rac1 did not affect the IFN-{gamma}-induced phosphorylation of JAK1 (Fig. 5, B and C). These results imply that IFN-{gamma} activates Rac1 in a JAK-dependent manner. In this regard, it is possible that IFN-{gamma} activates JAK and then a guanine nucleotide exchange factor, thus leading to activation of Rac1 in astrocytes. The detailed mechanism will be clarified in the future studies.

Our findings raised the question that Rac1 plays a role in IFN-{gamma}-induced inflammatory signaling. In an effort to examine the role of Rac1 in IFN-{gamma} signaling, we used the well-characterized Rac1 mutant, RacN17, which lacks GTPase activity. IFN-{gamma}-enhanced GAS promoter activity was significantly attenuated in C6 astroglioma cells expressing RacN17, and similar effects were observed in primary rat astrocytes (Figs. 2 and 3). However, transient transfection with the constitutively active mutant, RacV12, did not cause a significant change in the GAS activity. These findings suggest Rac1 may participate as a mediator in IFN-{gamma}-signaling and that the endogenous expression level of Rac1 may be sufficient to perform this role in rat astrocytes. The contribution of Rac1 in IFN-{gamma}-inducible transcriptional regulation was further demonstrated by measuring the transcription of MCP-1 and ICAM-1, which have STAT-binding motifs in their promoter regions (25, 26). The basal level of these transcripts in cells expressing RacN17 was significantly lower than in control cells. Moreover, the induction of these transcripts by IFN-{gamma} in cells expressing RacN17 was markedly weaker compared with control cells (Fig. 3B). Consistent with these results, cell surface expression of ICAM was also apparently low down in cells expressing RacN17 (Fig. 3C). In addition, the results in the present study showed that Rac1 deficiency attenuated IFN-{gamma}-stimulated tyrosine phosphorylation of not only STAT1 but also STAT3. However, it did not reduce the expression level of IFN-{gamma}R{alpha} and the phosphorylated level of JAK1 (Figs. 4 and 5). The phosphorylation of JAK1 in C6-RacN17 was rather elevated and sustained compared with that in C6 control cells (Fig. 5B). One possible explanation for this is decrease of IFN-{gamma}-induced inhibitory molecules for JAK1, such as suppressors of cytokine signaling (SOCS), in C6-RacN17 cells. To prevent detrimental effects, the intensity and duration of JAK-STAT activation are tightly regulated. Generally, SOCS are present in cells at very low levels, but are rapidly transcribed after exposure of cells to stimulus. SOCS has been reported to bind to the JAKs and inhibits their tyrosine kinase activity (35, 36). That is, Rac1 deficiency may reduce the phosphorylation of STATs, and then diminish the induction of IFN-{gamma}-induced inhibitory molecules for JAK1. Consequently, reduced action of JAK1 inhibitors may lead to sustained phosphorylation of JAK1 in C6-RacN17 cells. Taken together, these findings convincingly show that Rac1 contributes to IFN-{gamma} signaling at least at the level of STAT phosphorylation.

Next question is how Rac1 regulates STAT phosphorylation. We considered the possibility that Rac1 could interact with IFN-{gamma}R complex and augment its activity. It has been reported that Rac1 directly bind to several proteins, including p67phox, p21-activated kinase 1, and STAT3, and then stimulate their activity by constitution of active complex (15, 37). The results in this study showed several interesting findings about the roles of Rac1 in IFN-{gamma} signaling. First, Rac1 was coimmunoprecipitated with IFN-{gamma}R complex, including IFN-{gamma}R{alpha}, STAT1, and STAT3. Second, the association of Rac1 with IFN-{gamma}R{alpha} was detected in the absence of IFN-{gamma}. Third, the association of Rac1 with IFN-{gamma}R{alpha} was also detected in C6-RacN17 cells as well as C6 cells. Fourth, IFN-{gamma} significantly increased the association of Rac1 with either STAT1 or STAT3 in C6 cells while not in C6-RacN17 cells. These results suggest that Rac1 may augment the formation of active IFN-{gamma}R complex including IFN-{gamma}R{alpha} and STATs in response to IFN-{gamma}, thereby contributing to maximal activation of STAT1 and STAT3. Previously, Simon et al. (15) suggested that Rac1 could directly bind to STAT3 by growth factors and regulate STAT3 phosphorylation in COS-1 cells. However, interaction between Rac1 and IFN-{gamma}R{alpha} has not yet been reported. Although intensive experiments are required for explaining the questions about the association of Rac1 with IFN-{gamma}R complex, our findings suggest a novel cross-talk between Rac1 and IFN-{gamma}-induced inflammatory signaling in rat astrocytes. In summary, the present study suggests that Rac1 is rapidly activated by IFN-{gamma}, and then contributes to activation of STAT phosphorylation as an auxiliary mediator, thereby resulting in maximal activation of inflammatory signaling responses in rat astrocytes.


   Acknowledgments
 
We thank Dr. Alan Hall (University College London, London, U.K.) for providing pEXV-RacN17 and pEXV-RacV12, and Dr. Min-ho Shong (Chungnam National University, Daejon, Korea) for providing pGL-8 GAS plasmid.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the interdisciplinary research program of the Korean Science and Engineering Foundation (KOSEF 1999-2-207-004-5), KOSEF through the Brain Disease Research Center at Ajou University, and Grant M103KV010006 03K2201 00650 from the Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, Republic of Korea (to E.-H.J.). Back

2 E.J.P. and K.-A.J. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Eun-Hye Joe, Department of Pharmacology, School of Medicine, Ajou University, Suwon, 442-721, Korea. E-mail address: ehjoe{at}ajou.ac.kr Back

4 Abbreviations used in this paper: GAS, {gamma}-IFN-activated sequence; GPCR, G protein-coupled receptor; PAK, p21-activated kinase; PBD, p21-binding domain; SOCS, suppressor of cytokine signaling; Sos, son of sevenless. Back

Received for publication February 23, 2004. Accepted for publication August 10, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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